Subcellular mechanisms of subtype-specific neuron vulnerability in ALS and FTD: dysregulation of synapse-localized RNA, protein, and translation in mouse models and human cortico-spinal assembloids

ALS 和 FTD 中亚型特异性神经元脆弱性的亚细胞机制：小鼠模型和人类皮质脊髓组合体中突触定位 RNA、蛋白质和翻译的失调

• 批准号: 10716562
• 负责人: JEFFREY D MACKLIS
• 金额: $ 200.19万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716562
• 关键词: 3-Dimensional; Affect; Age; Amyotrophic Lateral Sclerosis; Area; Awareness; Axon; Biological Process; Brain; C9ORF72; Cells; Cerebral cortex; Cognition; Degenerative Disorder; Development; Elements; Emotions; Exhibits; Foundations; Frontotemporal Dementia; Functional disorder; Future; Genes; Genetic; Genetic Risk; Growth Cones; Human; Investigation; Knowledge; Label; Measures; Modeling; Molecular; Motor Neurons; Movement; Mus; Mutation; Neurons; Organoids; Output; Parents; Pathology; Pathway interactions; Patients; Population; Proteins; Proteome; Proteomics; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Sorting; Specificity; Spinal; Spinal Cord; Subcellular structure; Synapses; Synaptosomes; Testing; Therapeutic; Transcript; Transgenic Organisms; Translational Regulation; Translations; Untranslated RNA; Vertebral column; Wild Type Mouse; Work; comparative; familial amyotrophic lateral sclerosis; fluorophore; frontotemporal lobar dementia amyotrophic lateral sclerosis; genetic variant; induced pluripotent stem cell; insight; mosaic; motor deficit; mouse model; mutant; neuronal cell body; novel; novel strategies; particle; presynaptic neurons; prevent; protein TDP-43; ribosome profiling; risk variant; superoxide dismutase 1; synaptogenesis; trafficking; transcriptome; transcriptome sequencing

We propose to apply to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) newly developed subcellular approaches to investigate subtype-specific RNA, protein, and translational mechanisms of selective vulnerability to closely molecularly and developmentally related cerebral cortex neurons. ALS and FTD share genetics, and centrally involve degeneration of function-specific subtypes of subcerebral projection neruons (SCPN): in ALS, corticospinal neurons (CSN) controlling voluntary movement; in FTD, closely related von Economo neurons (VENs) and fork cells regulating emotion and cognition. Why these closely related neuronal subtypes are especially vulnerable in ALS and FTD is unknown and likely key to therapy, since in both patients and mice carrying variant genes of familial ALS or FTD (e.g. SOD1, TDP43, FUS, C9orf72), most neurons in the brain and most body cells express the variant risk gene, including many known RNA processing molecules, but only specific neuronal subtypes degenerate. Molecular differences between affected neuronal subtypes and other types of even closely related neurons might render affected neurons more vulnerable to dysfunction from mis-expression/mutation. By investigating vulnerable cortical populations and closely related unaffected populations, we aim to identify novel subcellular molecular specificities, abnormalities, or distinctly utilized pathways that render specific circuitry vulnerable to degeneration in ALS and FTD. This work will set a foundation toward future therapeutics to prevent/limit degeneration of specific cortical circuitry in ALS/FTD. We will quantitatively investigate subcellular RNA and protein localization (Aims 1, 3), and local translational regulation (Aims 2, 3), in subtype-specific synapses and somata of vulnerable CSN/SCPN, both in two complementary mouse models of ALS-FTD (hSOD1G93A Aims 1, 2; TDP-43Q331K Aim 3), and in refined human iPS cell-derived “assembloid” fusions of a cortical-like (Co-l) and a ventral spinal cord-like (vSC-l) organoid that exhibit remarkable neuron subset-specific projections and synapse formation from Co-l to vSC-l, modeling SCPN circuitry in ALS-FTD (Aim 4). We apply subtype-specific soma and synaptosome purification by FACS/novel small particle sorting with subtype-specific RNA-seq and new, ultra-low-input proteomics and ribosome profiling to interrogate ALS/FTD at the intersection of genetic risk, RNA processing, and subcellular specificity. We aim to identify subcellular molecular alterations in the affected circuitry, and to investigate whether these molecular alterations are specific to the ALS/FTD-affected subtypes by comparison with typically unaffected, but closely related cortical associative callosal projection neurons. This comprehensive “subtype-aware” analysis has promise to uniquely identify potential distinctions in subcellular (synapse- and soma-focused) RNA, protein, and/or translation as potential mechanisms of selective vulnerability. Together, this venturesome work will provide foundational molecular and subcellular insight into selective vulnerability of CSN/SCPN in ALS and FTD, merging new approaches across fields toward future therapy.

我们建议将肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）新 开发的亚细胞方法以研究亚型特异性RNA，蛋白质和翻译机制 选择性脆弱性，紧密分子和开发相关的脑皮质神经元。 Als和 FTD共享遗传学，并在中心涉及脑脑外投影功能特异性亚型的变性 Neruons（SCPN）：在ALS中，控制自愿运动的皮质脊髓神经元（CSN）；在FTD中，密切相关 冯经济神经元（Vens）和叉细胞调节情绪和认知。为什么这些密切相关 神经元亚型在ALS中尤其容易受到攻击，而FTD尚不清楚，可能是治疗的关键，因为 携带族或FTD的变异基因的患者和小鼠（例如SOD1，TDP43，FUS，C9ORF72），大多数 大脑和大多数身体细胞中的神经元表达变异风险基因，包括许多已知的RNA处理 分子，但仅特定的神经元亚型退化。受影响神经元之间的分子差异 亚型和其他类型的甚至密切相关的神经元可能会使受影响的神经元更容易受到影响 失误/突变功能障碍。通过调查脆弱的皮质种群并密切相关 不受影响的人群，我们旨在确定新型的亚细胞分子规格，异常或明显 利用使特定电路容易受到ALS和FTD变性的途径。这项工作将设定 基础，以防止ALS/FTD中特定皮质回路变性/限制/限制/限制。 我们将定量投资亚细胞RNA和蛋白质定位（目标1，3）和本地 翻译法规（AIM 2，3），亚型特异性突触和脆弱的CSN/SCPN的somata 在两个完整的ALS-FTD的鼠标模型中（HSOD1G93A AIMS 1，2; TDP-43Q331K AIM 3），在精制中 人IPS细胞衍生的类似皮质样（CO-L）和腹脊髓状（VSC-L）的“汇总”融合 裸露神经元亚集特异性项目和从共同至vsc-l的突触形成的类器官， 在ALS-FTD中对SCPN电路进行建模（AIM 4）。我们采用亚型特异性体和突触体纯化 通过FACS/新颖的小粒子分类，具有亚型特异性RNA-seq和新的，超低输入的蛋白质组学和 在遗传风险，RNA加工和亚细胞的交点处询问ALS/FTD的核糖体分析 特异性。我们旨在确定受影响电路中的亚细胞分子改变，并研究 这些分子改变是否特定于ALS/FTD影响的亚型 通常不受影响但密切相关的皮质关联callosal投射神经元。这个全面 “亚型意识”分析有望独特地识别亚细胞（突触和突触）中的潜在区别 以SOMA为中心的RNA，蛋白质和/或翻译作为选择性脆弱性的潜在机制。一起， 这项冒险的工作将为选择性提供基本的分子和亚细胞洞察力 CSN/SCPN在ALS和FTD中的脆弱性，将跨领域的新方法融合到未来的治疗中。